1. Field of the Invention
The present invention relates to slow release vehicles for delivering a biologically active agent. More particularly, the present invention relates to multivesicular liposomes containing IGF-I.
2. Description of Related Art
Proteins which are cleared rapidly from the circulation after intravenous or subcutaneous injections need to be administered repeatedly in order to maintain therapeutic blood levels. One of the proteins that needs frequent administration for therapeutic benefit is Insulin-like Growth Factor I (IGF-I). Mature, circulating IGF-I, a 7.65 kD protein, consists of B and A domains (homologous to the B and A chains of insulin). Unlike insulin, the B and A domains of the IGFs are connected by a C peptide, and contain an eight-amino-acid extension at the C-terminus, termed the D domain.
Native IGF-I contains three disulfide bonds involving the following residues: CysB6-CysB7, CysA6-CysA11, and CysB18-CysA20. Under reducing conditions, these disulfide bonds are broken and can become "scrambled" during oxidative refolding in denaturant solutions to yield two alternative disulfide isomers with distinct tertiary structures (L. O. Narhi et al., Biochemistry 32/5214-5221, 1993).
IGF-I is known to have multiple biological activities. Those with therapeutic potential are its activity in reversing catabolism in states of starvation, severe illness, or injury, in enhancing wound healing and nerve regeneration, and in reducing insulin resistance in diabetics. IGF-I is also known to generally stimulate the growth and maintenance of nervous tissue, increase glucose uptake of cells, and stimulate renal function. Chronic malnutrition and poorly controlled diabetes in the young are associated with lower circulating IGF-I levels and growth retardation.
Optimal treatment with IGF-I may require that the drug level be maintained at a specified level for a prolonged period of time. For example, optimal treatment of insulin dependency in diabetics may require maintenance of a relatively high level of IGF-I for a period of several days.
For certain other therapeutic usages, for example treatment of malnutrition or osteoporosis, a low level of IGF-I released over a period of several days would be beneficial.
One approach which has been used to provide controlled release compositions for drug delivery is liposome encapsulation. Among the main types of liposomes, multivesicular liposomes (Kim, et al., Biochim. Biophys. Acta; 728:339-348, 1983), are uniquely different from unilamellar liposomes (Huang, Biochemistry; 8:334-352, 1969; Kim, et al., Biochim. Biophys. Acta; 646:1-10, 1981), multilamellar liposomes (Bangham, et al, J. Mol. Bio., 13:238-252, 1965), and stable plurilamellar liposomes (U.S. Pat. No. 4,522,803). In contrast to unilamellar liposomes, multivesicular liposomes contain multiple aqueous chambers. In contrast to multilamellar liposomes, the multiple aqueous chambers of multivesicular liposomes are non-concentric with membrane distributed as a network throughout.
In multivesicular liposomes the encapsulation efficiency of some small molecules, such as cytosine arabinoside, is relatively low, and the release rate of encapsulated molecules in biological fluids is faster than is therapeutically desirable unless the osmolarity of the first aqueous component is adjusted to control the rate of release. EP 0 280 503 B1 discloses coencapsulation of a hydrochlo ride such as hydrochloric acid, with an active agent to control the rate of release of the active agent. Further research, disclosed in WO 95/13796, has shown that the release rate of agents from multivesicular liposomes in human plasma can also be controlled by introduction of a non-hydrochloride acid into the aqueous solution in which the agent is dissolved prior to forming the multivesicular liposome. It is also known (WO 96/08253) to control the rate of release of active agents by introducing other types of solutes called "osmotic spacers" into the aqueous solution in which the active agent is dissolved prior to formation of the multivesicular liposomes.
In addition to the biologically active agent and acids or osmotic spacers intended to control the rate of release of the biologically active agent from the liposomes, it is common practice to coencapsulate with the active agent compounds that are intended to serve any of a number of helper functions. For instance, certain biologically active compounds retain activity only when kept at a particular pH. Thus acids or buffers are often necessarily encapsulated in addition to the active agent to control the pH of the drug environment. In other cases, a counterion is incorporated to enhance solubility of a biologically active agent that has low solubility.
Thus the need exists for new high load and low load slow release formulations of IGF-I having the bioactivity of free drug in a vehicle suitable as a slow release drug depot. Since IGF-I is an expensive drug, whether purified from biological samples or produced recombinantly, a need also exists to achieve these goals while keeping the encapsulation efficiency as high as possible to avoid waste of the expensive active agents.